TOWARD 3D HUMAN BRAIN-LIKE TISSUES FOR TARGETING DYSREGULATED SYNAPSE AND PROTEOSTASIS MECHANISMS IN AUTISM SPECTRUM DISORDER Autism spectrum disorder (ASD) is a neurodevelopmental disease affecting nearly 1/50 children in US with an estimated $268 billion in annual costs. The disease is characterized by significant behavioral abnormalities that are often devastating to the quality of life for these patients. Behavior is directly related to the underlying changes of the central nervous system (CNS) brain tissue. Alterations in synapse maintenance and proteostasis pathways are found across ASD model systems, implicating a potential unifying approach for treating ASD. However, our knowledge of these two processes in both healthy and diseased, human relevant conditions is still limited. To deepen our understanding of the interconnectedness between these two fundamental processes in humans, the present application seeks to bioengineer 3D, human brain-like tissues. In addition to providing insight into the biology underlying disease, these tissues can be utilized to develop novel approaches to target identified aberrant mechanisms. As an initial starting point into building 3D ASD brain-like models, we propose to begin with cells from donors with a well-studied monogenetic, ASD-related disorder, Fragile-X syndrome (FXS). This approach will allow us to validate our model with a disorder which results in well documented phenotypic features. Our models will incorporate induced pluripotent stem cell (iPSC) derived neurons, astrocytes, and microglia (iNeurons, iAstrocytes, iMicroglia) to recapitulate 3D cell-cell interactions important for synapse maintenance. As such, we propose the following two Specific Aims: Aim 1: Develop a long-term 3D culture system compatible with relevant CNS cell types and outcomes (tissue level functionality, synapse/dendritic spine analyses, and proteostasis pathway biochemical analyses). Aim 2: Compare FXS-iPSC-based neuronal cultures vs. ?healthy?, genetically corrected iPSC neuronal cultures with respect to synapse maintenance and proteostasis.